Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (M_r 120 000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843–850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450_BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450_BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its M_r of 38 000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH₂-terminal amino acid is proline; the penultimate NH₂-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450_BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450_BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450_BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450_BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.     

Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli

In a recent publication (Narhi, L.O. and Fulco, A.J.[1986] J. Biol. Chem. 261, 7160-7169) we described the characterization of a catalytically self-sufficient 119,000 Dalton cytochrome P-450 fatty acid monooxygenase (P-450BM-3) induced by barbiturates in Bacillus megaterium ATCC 14581. We have now examined cell-free preparations from 12 distinct strains of B. megaterium and from one or two strains each of B. alvei, B. brevis, B. cereus, B. licheniformis, B. macerans, B. pumilis and B. subtilis for the presence of this inducible enzyme. Using Western blot analyses in combination with assays for fatty acid hydroxylase activity and cytochrome P-450, we were able to show that 11 of the 12 B. megaterium strains contained not only a strongly pentobarbital-inducible fatty acid monooxygenase identical to or polymorphic with P-450BM-3 but also significant levels of two smaller P-450 cytochromes that were the same as or similar to cytochromes P-450BM-1 and P-450BM-2 originally found in ATCC 14581. Unlike the 119,000 Dalton P-450, however, the two smaller P-450s were generally easily detectable in cultures grown to stationary phase in the absence of barbiturates and, with some exceptions, were not strongly induced by pentobarbital. None of the non-megaterium species of Bacillus tested exhibited significant levels of either fatty acid monooxygenase activity or cytochrome P-450. The one strain of B. megaterium that lacked inducible P-450BM-3 was also negative for BM-1 and BM-2. However, this strain (ATCC 13368) did contain a small but significant level of another P-450 cytochrome that others have identified as the oxygenase component of a steroid 15-beta-hydroxylase system. Our evidence suggests that the BM series of P-450 cytochromes is encoded by chromosomal (rather than by plasmid) DNA.     

Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium

The gene encoding barbiturate-inducible cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH2-terminal sequence of P-450BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an Mr of 47,439. The sequence is most, but less than 27%, similar to that of P-450CAM from Pseudomonas putida, so that P-450BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH-P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987-10995).     

Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium

In a previous publication (Narhi, L. O. and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium. This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein. We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons. The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3. The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography. All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite. The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein. T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate. T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate. Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding. Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other. In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein.     

Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium

Cytochrome P-450BM-3 (P-450BM-3) from Bacillus megaterium incorporates both a P-450 and an NADPH:P-450 reductase in proteolytically separable domains of a single, 119-kDa polypeptide and functions as a fatty acid monooxygenase independently of any other protein. A 5-kilobase DNA fragment which contains the gene encoding P-450BM-3 was sequenced. A single continuous open reading frame starting at nucleotide 1541 of the 5-kilobase fragment correctly predicted the previously determined NH2-terminal protein sequences of the trypsin-generated P-450 and reductase domains and, in toto, predicted a mature polypeptide of 1,048-amino acid residues with Mr = 117,641. The trypsin site was found at arginine residue 471. The P-450 domain is most similar (about 25%) to the fatty acid omega-hydroxylases of P-450 family IV, while the reductase domain exhibits some 33% sequence similarity with the NADPH:P-450 reductases of mammalian liver. Both the P-450 and reductase domains of P-450BM-3 define new gene families but contain highly conserved segments which display as much as 50% sequence similarity with P-450s and reductases of eukaryotic origin. The mRNA for P-450BM-3 was found by S1 mapping to be 3,339 +/- 10 nucleotides in length. In the accompanying paper, two regions in the 1.5 kilobases 5' to the P-450BM-3 coding region have been implicated in the regulation of P-450BM-3 gene expression.     

Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts

In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique. The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B. megaterium enzyme and contains the same N-terminal amino acid sequence. The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase. In E. coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital. However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B. megaterium via an E. coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital. This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA. The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B. megaterium but is absent in the E. coli clone.     

Purification and NH2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.     

The importance of serum lipoproteins in the cytolytic action of 7 beta-hydroxycholesterol on cultured hepatoma cells

A soluble cytochrome P-450-dependent fatty acid monooxygenase activity obtained from Bacillus megaterium ATCC 14581 can be induced by at least 13 different barbiturates. In general, the potency of these compounds as inducers increases with their increasing lipophilicity. We have now shown that at least 4 of these barbiturates (phenobarbital, secobarbital, pentobarbital and methohexital) seem to induce the same active cytochrome P-450-containing enzyme by a non-substrate type mechanism. The partially purified enzymes obtained from cultures induced with each of the 4 barbiturates tested were all of similar molecular size (Mr = 130,000 +/- 10,000) and had similar turnover numbers (1400-1800 +/- 300) with either palmitoleate or myristate as substrates. None of the tested barbiturates served as substrates, activators or inhibitors of any of the monooxygenase preparations, nor did they appear to interact in any way with the monooxygenase enzyme or the P-450 component.     

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).     

Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium.

Cell-free extracts from sonically disrupted Bacillus megaterium ATCC 13368 hydroxylated a variety of 3-oxo-delta4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic and 3beta-hydroxy-delta5-steroids did not serve as substrates for the 15beta-hydroxylase system. Using ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel ACA-54 it was possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system was fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin had an apparent sulfur to iron ration of 0.98 and showed g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic reso0 times and the preparation contained 1 to 2 nmol of cytochrome P-450 per mg of protein. This preparation of cytochrome P-450meg sedimented as a homogeneous zone on sucrose gradients with a sedimentation coefficient of 3.3 S and contained 0.94 nmol of heme per nmol of cytochrome P-450. The oxidized form of cytochrome P-450meg showed absolute absorption maxima at 416, 528, and 565 nm whereas the reduced form showed maxima at 411 and 542 nm. The following scheme is suggested for the electron transport in the 15beta-hydroxylase system in B. megaterium: NADPH leads to megaredoxin reductase leads to megaredoxin leads to cytochrome P-450meg.     

Isolation of a new form of cytochrome P-450 with prostaglandin A and fatty acid omega-hydroxylase activities from rabbit kidney cortex microsomes

Two different forms of cytochrome P-450, highly active in the omega-hydroxylation of prostaglandin A, and the omega- and (omega-1)-hydroxylation of fatty acids (P-450ka-1 and P-450ka-2), have been purified from kidney cortex microsomes of rabbits treated with di(2-ethylhexyl)-phthalate. On the basis of the peptide map patterns and NH2-terminal amino acid sequence, P-450ka-1 was determined to be a new form of omega-hydroxylase cytochrome P-450, whereas P-450ka-2 is identical to P-450ka reported earlier. The first 20 NH2-terminal amino acid sequence (ALNPTRLPGSLSGLLQVAGL) and (ALSPTRLPGSFSGFLQAAGL) of P-450ka-1 and P-450ka-2 showed 90 and 80% homology with that of the lung prostaglandin omega-hydroxylase, respectively, suggesting that these three cytochromes P-450 are members of the same omega-hydroxylase cytochrome P-450 gene family.     

Purification and characterization of rat pulmonary cytochrome P-450

The pulmonary cytochrome P-450, P450 L-2, was purified 460-fold from pulmonary microsomes of untreated male rats. Its specific content was 10.6 nmol/mg of protein. The monomeric molecular weight was 54,000 on SDS-polyacrylamide gel electrophoresis. The CO-reduced absorption maximum of P450 L-2 was at 451 nm, and the oxidized heme iron appeared to be in the low-spin state, as deduced from the Soret maximum at 421 nm. P450 L-2 had high lauric acid omega- and (omega-1)-hydroxylation activities, but low prostaglandin A1 omega- and (omega-1)-hydroxylation activities. It catalyzed the O-dealkylation of 7-ethoxycoumarin, but was not efficient in the hydroxylation of testosterone or the N-demethylation of aminopyrine. The NH2-terminal amino acid sequence of P450 L-2 was V-L-N-F-L-X-P-X-L (X being an unidentified residue). The catalytic properties of P450 L-2 resembled those of P450 K-5, the major rat renal cytochrome P-450. However, anti-P450 K-5 antibody did not cross-react with P450 L-2, and these forms had different NH2-terminal sequences. To judge from the results of NH2-terminal sequence analysis, P450 L-2 seems to be placed in the IVB gene family. Also, P-450 IIB1 was detected by immunoblotting in one of the peaks on ion-exchange HPLC during the purification of P450 L-2, suggesting the presence of P-450 IIB1 in rat pulmonary microsomes.     

Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium

A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.     

Separation of two constitutive forms of cytochrome P-450 active in aminopyrine N-demethylation from rabbit intestinal mucosa microsomes

Two constitutive forms of cytochrome P-450, designated P-450ib and P-450ic, were purified from intestinal mucosa microsomes of untreated rabbits. P-450ib and P-450ic have minimal molecular weights of 56 000 and 49 000, respectively, as determined by calibrated sodium dodecyl sulphate polyacrylamide gel electrophoresis. The CO-reduced difference spectral maximum of cytochrome P-450ib is at 450 nm and P-450ic is at 451 nm. Both the cytochromes preferentially demethylate aminopyrine, benzphetamine and N,N-dimethylaniline in the presence of NADPH-cytochrome P-450 reductase. Cytochrome P-450ib has absorption maxima at 417, 535 and 573 nm in the oxidized form, indicating that this cytochrome is in a low-spin state. Ouchterlony double-diffusion studies show that cytochrome P-450ib does not cross-react with antisera against liver cytochrome P-450LM2 purified from phenobarbital-treated rabbits, but P-450ic cross-reacts with spur formation. Unlike cytochrome P-450ib, P-450ic is very similar, if not identical, to liver cytochrome P-450LM2 on the basis of its molecular weight, spectral properties, catalytic activities and immunochemical properties.     

Reconstitution of the fatty acid hydroxylation function of cytochrome P-450BM-3 utilizing its individual recombinant hemo- and flavoprotein domains.

Cytochrome P-450BM-3 is a catalytically self-sufficient fatty acid omega-hydroxylase with two domains. Functional and primary structure analyses of the hemo- and flavoprotein domains of cytochrome P-450BM-3 and the corresponding microsomal cytochrome P-450 system have shown that these proteins are highly homologous. Prior attempts to reconstitute the fatty acid hydroxylation function of cytochrome P-450BM-3, utilizing the two domains, obtained either by trypsinolysis or by recombinant methods, were unsuccessful. In this paper, we describe the reconstitution of the fatty acid hydroxylation activity of cytochrome P-450BM-3 utilizing the recombinantly produced flavoprotein domain (Oster, T., Boddupalli, S. S., and Peterson, J. A. (1991) J. Biol. Chem. 266, 22718-22725) and its hemoprotein counterpart. The rate of fatty acid-dependent oxygen consumption was shown to be linear when increasing concentrations of the hemoprotein domain are added to a fixed concentration of the flavoprotein domain and vice versa. The combination of the hemo- and flavoprotein domains in a ratio of 20:1 respectively, in the reaction mixture, results in the transfer of 80% of the reducing equivalents from NADPH for the hydroxylation of palmitate at 25 degrees C. The ratio of the regioisomeric products obtained for lauric, myristic, and palmitic acids was similar to that obtained with the holoenzyme form of cytochrome P-450BM-3. The reconstitution of the fatty acid omega-hydroxylase activity, using the soluble domains of cytochrome P-450BM-3, without added factors such as lipids, may be useful for structure/function comparisons to their eukaryotic counterparts.     

Purification and characterization of two forms of cytochrome P-450 with omega-hydroxylase activities toward prostaglandin A and fatty acids from rabbit liver microsomes

Two forms of cytochrome P-450 (P-450), designated as P-450LPGA omega 1 and P-450LPGA omega 2, have been purified to specific contents of 17.9 and 11.1 nmol P-450/mg protein, respectively, from liver microsomes of rabbits treated with di(2-ethylhexyl)phthalate (DEHP), a peroxisomal proliferator. The purified P-450LPGA omega 1 and P-450LPGA omega 2 were found to have apparent molecular weights of 52,500 and 53,000, respectively. They showed absorption maxima at 451 and 450 nm in the carbon monoxide-difference spectra for their reduced forms, respectively. The two P-450s both efficiently catalyzed the omega-hydroxylation of prostaglandins A1 (PGA1) and A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate. In a reconstituted system, various metal ions such as Na+ and Mg2+ stimulated these reactions. The P-450s exhibited no detectable activity toward several xenobiotics tested. The two P-450s showed different peptide map patterns following limited proteolysis with Staphylococcus aureus V8 protease or papain. The NH2-terminal amino acid sequences (ALNPTRLPGSLSGLLQVAGL and ALSLTRLPGSFSGFLQAxGLLGLLL) of P-450LPGA omega 1 and P-450LPGA omega 2 were identical at 18/20 and 19/24 positions with that of the lung prostaglandin omega-hydroxylase from pregnant rabbits, respectively. An antibody against P-450LPGA omega 2 recognized a 52,000-53,000 molecular weight protein(s) in rabbit liver microsomes. The intensity of the immunoblot was significantly increased in liver microsomes from rabbits treated with DEHP, but not with phenobarbital or 3-methylcholanthrene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laboratory evolution of a soluble,self-sufficient,highly active alkane hydroxylase

We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.     

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.     

Characterization of recombinant Bacillus megaterium cytochrome P-450 BM-3 and its two functional domains

Bacillus megaterium cytochrome P-450BM-3 and its two functional domains, the heme and flavin domains, have been purified and characterized using an Escherichia coli expression system. Recombinant P-450BM-3 behaves both spectrally and enzymatically the same as the enzyme produced from the natural host, B. megaterium, and another E. coli system recently described (Bouddupalli, S. S., Estabrook, R. W., and Peterson, J. A. (1990) J. Biol. Chem. 265, 4233-4239). Reduction of the flavins in P-450BM-3 domain with NADPH appears to be very similar to microsomal P-450 reductases where two reducing equivalents are consumed to fully reduce the FMN while the FAD is converted to the semiquinone in an one electron reduction. NADPH reduction of the heme occurs only in the presence of substrate suggesting, by analogy with the cytochrome P-450CAM system, a possible increase in iron redox potential of the heme upon substrate binding which facilitates electron transfer from the flavins to the heme. The flavin domain retains a high level of cytochrome c reductase activity and also reacts with NADPH to give a 3-electron reduced product. The heme domain retains the ability to bind substrate and generates the characteristic 450-nm absorption band upon reduction in the presence of CO. The heme domain has been crystallized and a preliminary set of x-ray diffraction data obtained.